The present invention relates to novel compounds and their use in therapy (e.g. cancer therapy) and diagnosis. More particularly, the present invention relates to novel inhibitors of epidermal growth factor receptor tyrosine kinase (EGFR-TK) and their application in the treatment of EGFR-TK related diseases and disorders (e.g. cancer), and to novel radiolabeled inhibitors of EGFR-TK and their application as bioprobes for, e.g., Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT), and as radiopharmaceuticals for radiotherapy. The present invention further relates to novel processes of preparing and optionally radiolabeling the EGFR-TK inhibitors.
The presently used anticancer therapy is mostly based on non-specific cytotoxic agents, such as cisplatin, paclitaxel, doxorubicin, topotecan and 5-fluorouracil (5-FU). These cytotoxic agents are mainly directed at inducing DNA damage, inhibiting DNA synthesis or disrupting the cytoskeleton. The toxicity of these agents limits their dosage quantities, which often results in the disease recurrence. In some cases, the maximum tolerated dose is even below the minimum effective dose for tumor regression (Ciardiello, 2000; Renhowe, 2001; Rowinsky, 2000).
The realization that cancer cells differ from normal cells in their aberrant signal transduction has given impetus to cancer researchers to target the cancer cells while searching for cancer therapy and more recently for cancer diagnosis.
Polypeptides such as growth factors, differentiation factors, and hormones often mediate their pleiotropic actions by binding to and activating cell surface receptors with an intrinsic intracellular protein tyrosine kinase activity.
The Epidermal Growth Factor Receptor (EGFR/Her-1/) belongs to the ErbB receptor family involved in proliferation and differentiation of normal and malignant cells (Artega et al., 2001). Overexpression of EGFR and its enhanced signaling are a frequent hallmark of human epithelial cancers, and it contributes to the initiation, progression and/or invasiveness of human cancers (Tokunaga et al., 1995; Shimada et al., 1996; James et al. 2004; Levitzki et al., 2003). Overexpression of Epidermal Growth Factor Receptor (EGFR) is present in at least 70% of human cancers (Seymour, 2001) such as non-small cell lung carcinomas (NSCLC), breast cancers, gliomas, squamous cell carcinoma of the head and neck, and prostate cancer (Raymond et al., 2000, Salomon et al., 1995, Voldborg et al., 1997). Furthermore, correlation between EGFR overexpression and metastasis formation, therapy resistance, poor prognosis and short survival have been recently described (Tokunaga et al., 1995; Shimada et al., 1996, Rae and Lippman, 2004, and Levitzki 2003). As a result, EGFR-TK has become a major target for the development of specific anticancer drugs.
Examples of such FDA approved therapies include reversible EGFR-TK inhibitors, such as gefitinib (Iressa™, ZD1839; AstraZeneca, Wilmington, Pa.) for treatment of locally advanced or metastatic chemotherapy refractory NSCLC and erlotinib (Tarceva™; Genentech, San Francisco, Calif.) for treating locally advanced or metastatic chemotherapy refractory NSCLC and, in addition to gemcitabine, as a first choice treatment of locally advanced, inoperable or metastatic pancreatic cancer. Lapatinib (GW572016, GlaxoSmithkline) and PKI-166 both are under phase III clinical trials.
Additional anti-EGFR targeted therapies, currently under clinical trials, include, for example, the irreversible inhibitor CI-1033.
Compounds belonging to the 4-Anilinoquinazolines family, which are also referred to herein as 4-(phenylamino)quinazolines, have also been shown to potently and selectively inhibit EGFR-TK activity by binding reversibly to an inner membrane ATP binding site on EGFR-TK, (Faaland et al., 1991; Miyaji et al., 1994; Gazit et al., 1996; Artega et al., 1997; Nelson and Fry, 1997; Johnstrom et al., 1997; Smaill et al., 1999; Tsou et al., 2001; and Han et al., 1996), the prototype for such compounds being the small molecule AG 1478, also known as PD 153035 (Fry et al., 1994; Levitzki and Gazit, 1995), which is presently in clinical development. The FDA approved Iressa described above also belongs to this quinazoline family (Baselga and Averbuch, 2000).
While the above-described agents are reversible EGFR-TK inhibitors, their potency is limited by non-specific binding and rapid blood clearance. Thus, irreversible EGFR-TK inhibitors, which are based on the structure of AG 1478, have been proposed (Fry et al., 1998; Smaill et al., 2000; and U.S. Pat. Nos. 6,153,617 and 6,127,374). PD168393 and PD160678 are representative examples of such irreversible inhibitors. The irreversible binding of these inhibitors was achieved by substituting the 6 or 7 position of the quinazoline ring of an 4-(anilino)quinazoline derivative with an α,β-unsaturated carboxylic group, preferably an acrylamide group, which binds covalently to the Cys-773 at the EGFR-TK ATP binding site. Some of these compounds showed high potency toward EGFR inhibition in both in vitro and in vivo experiments (Smaill et al., 2000). However, as is detailed hereinunder, more recent studies showed that these irreversible EGFR-TK inhibitors are limited by a relatively low accumulation at EGFR-expressing tumor cells.
Hence, it would be highly advantageous to have irreversible EGFR-TK inhibitors with improved efficacy, which could serve as potent anticancer agents.
In addition to the growing efforts for targeting and inhibiting the EGFR in cancerous cells, the role that EGFR overexpression plays in cancer development is gradually unraveled. Consequently, there has been a growing interest in the use of EGFR-TK inhibitors as radiotracers for molecular imaging of EGFR overexpressing tumors via nuclear medicine modality such as Positron Emission Tomography (PET).
The use of radioactive nuclides for medicinal purposes is well known in the art. Biologically active compounds that bind to specific cell surface receptors or that otherwise modify cellular functions have received some consideration as radiopharmaceuticals, and therefore, when labeled with a radioactive nuclide, such compounds are used as biospecific agents in radioimaging and radiotherapy.
Positron Emission Tomography (PET), a nuclear medicine imagine technology which allows the three-dimensional, quantitative determination of the distribution of radioactivity within the human body, is becoming an increasingly important tool for the measurement of physiological, biochemical, and pharmacological function at a molecular level, both in healthy and pathological states. PET requires the administration to a subject of a molecule labeled with a positron-emitting. nuclide (radiotracer) such as 15O, 13N, 11C, and 18F, which have half-lives of 2, 10, 20, and 110 minutes, respectively.
Single Photon Emission Computed Tomography (SPECT) is a form of chemical imaging in which emissions from radioactive compounds, labeled with gamma-emitting radionuclides, are used to create cross-sectional images of radioactivity distribution in vivo. SPECT requires the administration to a subject of a molecule labeled with a gamma-emitting nuclide such as 99mTc, 67Ga, 111In and 123I.
The use of nuclear medicine imaging techniques such as Single Photon Emission Compute Tomography (SPECT) and Positron Emission Tomography (PET), along with a suitable radiotracer that binds to EGFR irreversibly, can therefore provide for in vivo drug development and identification of a lead chemical structure to be used as an EGFR-TK biospecific agent for radiotherapy or as a labeled bioprobe for diagnosis by radioimaging. Nuclear imaging can be further used for in vivo mapping and quantification of the receptor-kinase in cancer. Using a labeled EGFR-TK irreversible inhibitor would enable both the identification of patients having tumors overexpressing EGFR, and the study of changes in the levels of EGFR expression during therapy. Such a diagnostic method can lead to a better patient management and differentiation in regards to therapeutic course of action. Moreover, the increasing demand to incorporate diagnostic methods into clinical studies of EGFR-targeted therapies suggests a potential future use of EGFR-labeled inhibitors.
Radiolabeling of 4-anilinoquinazoline EGFR-TK inhibitors has been reported in the art. For example, a radioiodinated analog of PD 153035 and in vitro binding studies therewith in MDA-486 cells have been reported (Mulholland et al., 1995). PD 153035 labeled with carbon-11 in the 6,7-methoxy groups has been evaluated in rats implanted with human neuroblastoma xenografts (SH-SY5Y) but specific uptake was not determined in a blocking study (Johnstrom et al, 1998). PD 153035 was also labeled with carbon-11 specifically at the 7-methoxy position and biodistribution experiments were performed in normal mice, but uptake specificity could not be demonstrated as administration of an enzyme-blocking dose of PD 153035 caused an increase in tracer uptake in the tissues studied (Mulholland et al., 1997). The same abstract reported the labeling of the 7-(2-fluoroethoxy) PD 153035 analog with fluorine-18, but no biological experiments with this tracer were described.
U.S. Pat. No. 6,126,917 (to the present assignee), which is incorporated by reference as if fully set forth herein, Mishani et al., 1999 and Bonasera et al., 2000, all teach reversible inhibitors of EGFR-TK of the 4-anilinoquinazoline family labeled with fluorine-18 on the aniline ring. These compounds were tested in vitro, in vivo and by PET image analysis. While some of these compounds showed effective (reversible) inhibition activity in vitro, they were found to exhibit limited efficiency as tracers for the imaging of EGFR-TK in vivo due to kinetic factors such as kon and koff and rapid blood clearance, as was further demonstrated by an animal PET comparative study between fluorine-18 FDG and these radiolabeled compounds. It is assumed that the discrepancy between the encouraging in vitro results and the discouraging in vivo results derives from the ATP competition at the compounds' binding site.
In order to eliminate this ATP binding competition and thus obtain a better specificity and inhibitory effect of radiolabeled EGFR-TK inhibitors, which would potentially result in higher diagnostic performance and high radiotherapeutic activity in tumor cells expressing EGFR-TK, radiolabeled irreversible inhibitors, based on those described by Smaill et al. (Smaill et al., 2000), were synthesized.
As is taught in U.S. Pat. No. 6,562,319 (to the present assignee), which is incorporated by reference as if fully set forth herein, and in Ben David et al., 2003, acrylamido derivatives of 4-anilinoquinazoline were synthesized, radiolabeled by 11C and tested for PET imaging of tumor cells overexpressing EGFR-TK. Indeed, these compounds showed irreversible and fast binding effect toward EGFR in in vitro studies conducted with A431 cells. However, while the ATP binding competition was eliminated and long-term inhibitory effect was obtained with these compounds in vitro, the in vivo studies in tumor bearing rats did not indicate high accumulation of the compounds in the tumor. In further in vivo studies, fast decomposition and clearance, as well as high accumulation of the compounds in the intestine, were observed, suggesting that the performance of this class of compounds is limited by low in vivo bioavailability and degradation.
Therefore, further studies have focused on the design and development of novel derivatives of irreversible inhibitors as PET imaging agent candidates (Mishani et al., 2004). U.S. patent application Ser. No. 10/659,747 (Publication No. 2004/0265228, recently granted), which is incorporated by reference as if fully set forth herein, discloses, for example, a novel group of compounds, the 4-dimethylamino-but-2-enoicacid [4-(phenylamino)-quinazoline-6-yl]-amides. These compounds held a favorable profile, characterized by a remarkable inhibitory potency toward the EGFR, elevated chemical and biological stabilities and sufficient selectivity with respect to other tested tyrosine kinase receptors. The lead compound of this group, referred to as ML04 (see, FIG. 1a), was labeled with 11C and 18F, and its potential as EGFR PET imaging agent was evaluated. However, these irreversible compounds exhibited insufficient bioavailability, characterized by low circulating blood levels after oral administration. This limited performance was attributed to the low solubility of these compounds under physiological conditions and to rapid metabolic pathway caused by the chemical reactivity of the acrylamide and butynamide unsaturated bonds.
In International Patent Application WO 04/064718, which is incorporated by reference as if fully set forth herein, a novel class of irreversible EGFR-TK inhibitors characterized by reduced biodegradation, enhanced bioavailability and hence by improved in vivo performance as compared with the structurally related reversible and irreversible EGFR-TK inhibitors described above, has been disclosed. The compounds belonging to this newly designed class have a leaving group such as α-chloroacetamide or an α-methoxyacetamide group attached to the quinazoline ring. According to the teachings of WO 04/064718, it was found that replacing the α,β-unsaturated side chain of the highly reactive carboxylic moiety, by the less reactive chloro and methoxy groups, which can further act as leaving groups and thus readily react so as to form a covalent bond with the cysteine moiety at the receptor binding site, resulted in potent irreversible inhibitors with enhanced biostability and bioavailability. It was thus found that such newly designed compounds, having an α-chloroacetamide or an α-methoxyacetamide group attached to the quinazoline ring, show high affinity toward EGFR and high ability to irreversibly bind to the receptor, thus indicating their potential as improved EGFR-TK irreversible inhibitors and as a result as improved diagnostic and therapeutic agents. A representative member of this family of irreversible EGFR inhibitors is referred to herein as ML05 (see, FIG. 1b). Nonetheless, the use of these compounds remained limited due to insufficient solubility and biological stability thereof.
There is thus a widely recognized need for, and it would be highly advantageous to have, novel radiolabeled and non-radiolabeled inhibitors of EGFR-TK, devoid of the above limitations.
One common way to increase the blood-residency of proteins is by conjugating the proteins to a non-proteinaceous substance such as polyethylene glycol (PEG). Conjugation of PEG to proteins results in increased molecular size and stearic hindrance of the protein and, as a result, often improves the plasma half-lives and proteolytic-stability of the proteins, and decreases their immunogenicity and hepatic uptake (Chaffee et al., 1992; Pyatak et al., 1980). Conjugation of PEG further increases the solubility of proteins in body fluids.
The prior art, however, fails to teach or suggest conjugation of PEG to EGFR-TK inhibitors such as those described hereinabove.